Herpes simplex virus (HSV) causes lifelong latent infections in humans. It is responsible for significant disease, ranging from cold sores and genital infections to blindness and fatal encephalitis. The long- term objective of this project isto develop a novel intervention strategy to treat HSV infections. Despite much effort, there is no effective HSV vaccine. Resistance to antiviral drugs, such as acyclovir, contributes significantly to serious HSV disease and mortality. New, effective anti- herpesviral drugs are needed, and virus entry is a highly desirable target. HSV entry requires host 26S proteasome activity for intracellular transport of the entering capsid to the nuclear periphery. The pharmacodynamics of proteasome inhibitors that block herpesvirus infection has not been investigated. The proteasome inhibitor bortezomib is a clinically effective anti-neoplastic drug that is FDA-approved for treatment of hematologic malignancies. We will implement pilot studies to re- purpose bortezomib as an anti-herpesviral agent. Our Specific Aim is to determine whether bortezomib is effective at inhibiting HSV infection of pathophysiologically relevant cells in vitro and to elucidate its mechanism of action. To accomplish this, we propose the following sub-aims: (a) To delineate the pharmacodynamics of bortezomib-mediated inhibition of HSV-1 and HSV-2 infection, including determination of selectivity index; (b) To determine the effect of bortezomib on clinical isolates and drug-resistant strains of HSV; and (c) To ascertain the mechanism of bortezomib inhibition of HSV infection. We will assess the effect of bortezomib on virus entry by measuring incoming capsid transport and on proteasome-mediated disruption of host nuclear domain 10 (ND10) structures. Our experimental design employs techniques of cell biology, biochemistry, pharmacology, and molecular virology. Completing this pilot study will lay the critical groundwork for re-purposing an anti-cancer drug to treat HSV infections.